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In multiple myeloma, the overexpression of receptor activator of nuclear factor kappa B (NF-kappaB) ligand (RANKL) leads to the induction of NF-kappaB and activator protein-1 (AP-1)-related osteoclast activation and enhanced bone resorption. The purpose of this study was to examine the molecular and functional effects of proteasome inhibition in RANKL-induced osteoclastogenesis. Furthermore, we aimed to compare the outcome of proteasome versus selective NF-kappaB inhibition using bortezomib (PS-341) and I-kappaB kinase inhibitor PS-1145. Primary human osteoclasts were derived from CD14+ precursors in presence of RANKL and macrophage colony-stimulating factor (M-CSF). Both bortezomib and PS-1145 inhibited osteoclast differentiation in a dose- and time-dependent manner and furthermore, the bone resorption activity of osteoclasts. The mechanisms of action involved in early osteoclast differentiation were found to be related to the inhibition of p38 mitogen-activated protein kinase pathways, whereas the later phase of differentiation and activation occurred due to inhibition of p38, AP-1 and NF-kappaB activation. The AP-1 blockade contributed to significant reduction of osteoclastic vascular endothelial growth factor production. In conclusion, our data demonstrate that proteasomal inhibition should be considered as a novel therapeutic option of cancer-induced lytic bone disease.

The proteasome is a proteolytic complex for intracellular degradation of ubiquitinated proteins which are involved in cell-cycle regulation and apoptosis. A constitutively increased proteasome activity has been found in myeloma cells. We studied circulating proteasome levels and their prognostic significance in sera of 50 control subjects, 20 persons with monoclonal gammopathies of undetermined significance (MGUS), and 141 previously untreated patients with multiple myeloma (MM) by an anti-20S proteasome enzyme-linked immunoabsorbent assay (ELISA). Serum proteasome concentrations were significantly elevated in MM compared with controls (P < .001), in MM versus MGUS (P = .03), and in active (n = 101) versus smoldering (n = 40) MM (P < .001). In patients with active MM, there was a significant (P < .001) decrease from pretreatment to post-treatment proteasome concentrations in responders to chemotherapy, but not in nonresponders. Circulating proteasome levels were identified as a prognostic factor for overall survival in the univariate (P < .001 log-rank test) and in the multivariate (hazard ratio, 4.38) survival analysis in patients with active MM. We demonstrate for the first time that increased serum proteasome concentrations correlate with advanced disease and are an independent prognostic factor in MM.

Chronic myeloid leukemia developing after transplantation of solid organs and concomitant immunosuppression is a rare but still significant clinical phenomenon. We here describe an additional case of a 62-year-old male patient developing CML after orthotopic heart transplantation and medication with cyclosporine A, mofetil-mycophenolate, and steroids. Initial antileukemic therapy was imatinib at a standard dose and within 15 months of therapy a complete cytogenetic response was noted. In this report we discuss the clinical implications of these rare but biologically important cases.

Multiple myeloma (MM) is a malignancy with excessive production of monoclonal proteins. At disease presentation 30% of MM patients have significant renal impairment which may progress to renal failure requiring dialysis. Besides chemotherapy extracorporeal elimination procedures such as plasma exchange have been applied as adjuvant strategies to eliminate free light chains from circulating blood, however the efficacy was poor with older techniques. We report about a highly efficient method to eliminate serum free light chain (sFLC) using a newly designed protein leaking membrane in patients suffering from sFLC induced acute renal failure. The protein leaking membrane (HCO 1100) is characterized by increased pore size facilitating elimination of middle molecules such as sFLC kappa (22.5 kD). The HCO 1100 membrane was applied in a hemodialysis and hemodiafiltration mode and compared to standard procedures (high flux hemodialysis, hemodiafiltration and plasma exchange). Hemodiafiltration with the protein leaking membrane HCO 1100 was superior to all other extracorporeal replacement strategies in eliminating sFLC-kappa from circulating blood. A median blood reduction rate of 40.8% (range 13.9% - 66.4%) was achieved during hemodiafiltration. The corresponding peak clearance rate was 25 ml/min. Importantly, the poorest elimination rate was achieved by plasma exchange followed by standard high flux hemodialysis. Extracorporeal elimination strategies with the protein leaking membrane HCO 1100 may be a promising adjuvant treatment strategy for patients with sFLC nephropathy requiring dialysis. Hemodiafiltration and to lesser extend also hemodialysis with the HCO 1100 hemofilter are able to eliminate substantial amounts of sFLC kappa in MM patients.

Mantle cell lymphoma (MCL) is an incurable B cell lymphoma and novel treatment strategies are urgently needed. Proteasome inhibitors, e.g. bortezomib, act by targeting the catalytic 20S core of the proteasome and induce apoptosis in tumor cells. Among other mechanisms, they lead to cytoplasmic accumulation of the IκBa protein, resulting in a reduced NF-κB activity. Histone deacetylase inhibitors (HDAI), e.g. SAHA, promote histone acetylation, chromatin uncoiling, and transcription of a variety of genes. Previous studies have indicated that HDAIs also interfere with NF-κB signaling. Since NF-κB is constitutively activated MCL cells and plays a major role in a variety of cellular processes, we hypothesized synergist effects of bortezomib and HDAI in MCL cells. Human mantle cell lymphoma cell lines (JeKo-1 and Granta-519) were exposed to bortezomib and/ or SAHA for 4 to 48 hours. Cell viability and apoptosis were quantified by the MTT and annexin-V assay, respectively. The effect of the combination of both agents was analyzed using the median effect method of Chou and Talalay. Reactive oxygen species (ROS) were quantified by the fluorophore H2DCFDA. The functional role of ROS generation was assessed using the free radical scavenger N-acetyl-l-cysteine (LNAC). In addition, activated caspases, proteasome- and NF-κB activity were quantified. After 48 hours of incubation, IC50 of SAHA and bortezomib were noted at 0.8μM and 7.7nm in JeKo-1 cells and at 3.9μM and 5.7nm in Granta-519 cells, respectively. Combined incubation resulted in synergistic cytotoxic effects, as indicated by CI values

The ubiquitin-proteasome pathway was recently identified as a promising new therapeutic target in cancer treatment. An increased proteasome activity was described in certain cancer types, especially in multiple myeloma (MM) and also in mantle cell lymphoma (MCL). The proteasome inhibitor bortezomib has been approved for the treatment of refractory MM and has also shown reproducible activity in MCL. Recently we described a novel tripeptide compound, BSc2118, with inhibitory activity against all three proteolytic activities (post-glutamyl peptide hydrolase-like, trypsin-like and chymotrypsin-like activities) of the 20S proteasome (Cancer Res2006;66:7598–605). We investigated the in vitro effects of BSc2118 in MM and MCL cell lines by MTT cell viability and AnnexinV apoptosis-assays. Furthermore, the intracellular chymotrypsin-like proteasome activity and NF-κB activity were detected in MM and MCL cells by detection of proteasome-degraded peptides or NF-κB p65 subunit. In MM cell lines OPM-2 and U266 we could show a significant time and dose-dependent reduction of cell viability by BSc2118 with an IC50 at 48hrs of 52nM and 65nM, respectively, whereas the MM cell line RPMI-S was less sensitive with an IC50 of 287nM. Using AnnexinV assay, a dose-dependent induction of apoptosis by BSc2118 was shown after 48hrs incubation. Comparably, in MCL we also found a time and dose-dependent reduction of cell viability in the cell lines HBL-2, JeKo-1 and Granta-519 with an IC50 of 82nM, 130nM and 262nM, respectively. Furthermore, BSc2118 induced apoptosis in all three MCL cell lines. Additionally, we detected a significant dose-dependent inhibition of intracellular chymotrypsin-like proteasome activity in MM and MCL cells, and a dose-dependent inhibition of TNFα-induced NF-κB activation in the MM cell lines OPM-2 and RPMI-S. This is the first report of anti-tumor effects of the novel proteasome inhibitor BSc2118 in MM and MCL cells. The compound effectively reduces the cell survival and shows a high pro-apoptotic activity in the MM cell lines OPM-2 and U266 and a significant activity in MCL cell lines HBL-2 and JeKo-1. Mechanisms of action are the inhibition of proteasome and NF-κB activity. Since previous clinical trials have shown an activity of the proteasome inhibitor bortezomib in MM and MCL, and adverse effects of other proteasome inhibitors may differ, our preclinical data support the idea to consider BSc2118 as a promising new agent in anti-tumor drug development.

Myeloma bone disease is caused by an enhanced osteoclast activation and impaired osteoblast function. Until now, there is no specific treatment to restore osteoblast activity, and anti-myeloma therapies that lead to a disease remission are usually not associated with an increase of osteoblast markers. Recently, preclinical data suggested that proteasome inhibitors may enhance osteoblast function. Bortezomib (Velcade) represents the first substance from this group which is clinically used in relapsed multiple myeloma. To evaluate whether there is clinical evidence for an osteoblast stimulation under bortezomib treatment, we analyzed serum levels of two specific osteoblast markers, i.e. bone-specific alkaline phosphatase (BAP) and osteocalcin, in 25 multiple myeloma patients treated with bortezomib alone or in combination with dexamethasone. 56 percent of patients achieved a complete or partial remission. In the whole group of patients, mean serum levels of osteocalcin significantly increased from 6.3 μg/l before treatment to 10.8 μg/l after three months of therapy (P=0.024). In parallel, mean levels of BAP increased from 19.7 U/l to 30.2 U/l (P

Enhanced osteolytic bone resorption is a hallmark of multiple myeloma. Several studies demonstrated an elevation of RANKL levels in the bone marrow microenvironment in multiple myeloma, but serum levels of soluble RANKL (sRANKL) revealed controversial results. One study reported elevated sRANKL in serum in myeloma patients, but using the same test, the levels in the majority of the patients were below the detection limit in the hand of other groups. Thus, a novel test was developed which measures both soluble and OPG-bound RANKL (total-RANKL, tRANKL). The objective of the present study was to investigate the clinical significance of circulating levels of tRANKL in monoclonal gammopathies of undetermined significance (MGUS) or multiple myeloma (MM). Serum levels of tRANKL were analyzed by ELISA in 128 individuals: 20 healthy donors, 20 with MGUS and 88 newly diagnosed patients with multiple myeloma. Multiple myeloma patients had significantly (P